Nanosymposiums on Pain: Highlights From SfN 2017

The following is a report on two nanosymposiums on pain held at the 2017 Society for Neuroscience annual meeting, which took place November 11-15 in Washington, DC. See other reports on the meeting here and here.

Novel targets were a prominent theme at a nanosymposium on touch, itch, and pain held at the 2017 Society for Neuroscience annual meeting. Theodore (Ted) Price, University of Texas at Dallas, US, discussed one of those targets, PAR3, which belongs to the protease-activated receptor (PAR) family of G protein-coupled receptors (GPCRs) and has traditionally been thought to act as a co-receptor with other PARs. Thus far, most research on PARs, which are well-known pain mediators, has focused on other family members, particularly PAR2 (Ossovskaya and Bunnett, 2004).

Price and colleagues were investigating expression levels of various GPCRs in human and mouse dorsal root ganglia (DRG) and found that expression of PAR3 was much higher than other PARs such as PAR2. Furthermore, single cell RNA sequencing revealed that PAR3 was primarily expressed in nociceptors. This was confirmed by in situ hybridization on mouse DRG neurons, where PAR3 overlapped extensively with transient receptor potential receptor vanilloid type 1 (TRPV1) messenger RNA expression.

In order for PARs to be activated, a protease has to specifically cleave the amino terminal sequence of the receptor, which in turn exposes a new N-terminal sequence. This N-terminus functions as a tethered ligand that attaches to PAR within the binding pocket, thereby activating the receptor. To investigate PAR3 function the researchers designed several peptidomimetic ligands, derived from the PAR3 N-terminal sequence in humans, that stimulate calcium signaling in DRG and trigeminal ganglion neurons.

From this assay, they discovered a putative PAR3 agonist named C660. Injection of C660 directly into the hindpaw of mice caused mechanical hypersensitivity, which was absent in PAR3/PAR4 double knockout mice. Furthermore, C660 application alone and in combination with the sodium channel blocker tetrodotoxin to mouse spinal cord slices suggested that this might be a presynaptic physiological effect, according to electrophysiology experiments.

Together, the data suggest that PAR3 plays a novel role in promoting pain independent of other PARs, making it a novel target for pain treatment.

Rose Hill, University of California, Berkeley, US, presented results on a bioactive signaling lipid, sphingosine 1-phosphate (S1P), and its regulation of mechanical pain. S1P receptor 3 (S1PR3), a GPCR activated by S1P, was recently identified as a gene that is enriched in specialized subsets of mechanosensory neurons (Gerhold et al., 2013).

Hill used knockout mice that lacked S1PR3 (Kono et al., 2004) and showed that loss of this receptor decreased the animals’ sensitivity specifically to mechanical pain, as measured by von Frey or pinprick assays. The S1PR3 knockouts had no defects in light touch, proprioception, itch, or noxious heat detection.

To further investigate the role that S1P plays in mechanical pain in wild-type animals, Hill and colleagues locally administered TY 52156, a selective S1PR3 antagonist, or SKI II, a sphingosine kinase antagonist that decreases S1P levels, finding that the administration of either of these agents decreased the animals’ sensitivity to mechanical stimuli, similar to the knockout animals. The group also showed that direct administration of S1P to wild-type animals had no effect on mechanical sensitivity. The results suggest that pharmacological blockade of S1P/S1PR3 signaling in the periphery is sufficient to inhibit mechanical pain.

Meanwhile, in situ hybridization of mouse DRG to determine which subsets of sensory neurons expressed the S1PR3 gene pointed to high-threshold mechanoreceptors (HTMRs) that terminate as free nerve endings in the skin as a likely candidate.

In a collaborative effort, as part of a student project at the Marine Biology Laboratory Neurobiology Course, with Ellen Lumpkin, Ben Hoffman, and Stephanie Campos, Columbia University, US, the investigators then tested the response properties of these high-threshold A fiber mechanonociceptors (AM fibers) in a skin-nerve recording. AM fibers respond to mechanical force and are thinly myelinated fibers known for their free nerve endings. The group found that S1PR3 was required for responses to high-threshold mechanical stimuli. The overall firing rate of neurons from the S1PR3 knockout animals was decreased, and von Frey thresholds were significantly increased. Furthermore, knockout of S1PR3 significantly decreased the proportion of force-responsive adapting AM fibers, showing that S1PR3 is required for normal mechanosensitivity of those fibers.

To further understand how S1P regulates mechanonociceptors, the researchers performed whole-cell recordings in the presence or absence of S1P on neurons of S1PR3 reporter animals that express a functional S1PR3-mCherry fusion protein. S1P application to S1PR3-expressing mechanonociceptors significantly increased neuronal excitability and decreased tail currents in wild-type animals, as compared to the S1PR3 knockouts. In terms of downstream effects of S1PR3, when active, the receptor closed KCNQ2/3 potassium channels. KCNQ2 and KCNQ3 are potassium channels that are exclusively expressed in the nervous system, where they control neuronal excitability.

Overall, this work shows that S1P and S1PR3 are essential regulators of mechanical pain in somatosensory neurons. S1P activation of S1PR3 results in inhibition of KCNQ2/3 channels and constitutively promotes excitability of AM fibers and mechanical pain sensitivity. If, however, S1P production or S1PR3 itself is blocked, this opens up KCNQ2/3 channels, leading to decreased excitability and loss of mechanical pain sensitivity. Therefore, inhibitors that block the production of S1P or inhibitors of S1PR3 could be valuable targets for the development of new pain medications. (A preprint describing this work is available here).

Ashley Cowie, Medical College of Wisconsin, Milwaukee, US, is using optogenetics to understand the role of calcitonin gene-related peptide-α (CGRPα) neurons in neuropathic and inflammatory pain conditions. (Disclosure: the current writer works in the same lab as Cowie). The importance of these neurons after injury has been debated, since studies have used chemical ablation (McCoy et al., 2013), where entire populations of CGRPα neurons are lost. This could lead to compensatory effects, which is why optogenetics, a technique that provides better spatial and temporal precision, has gained popularity in pain studies.

Cowie presented results from her study using mice engineered to express the light-sensitive protein Archaerhodopsin-3 (Arch) specifically in CGRPα-expressing neurons. Findings indicated that CGRPα neurons were important for baseline thermal sensation, but not for baseline mechanosensation. However, after spared nerve injury (SNI), CGRPα neurons drove SNI-induced mechanical, heat, and cold hypersensitivity, which could be reversed by peripheral transient optogenetic inhibition of these sensory neurons. Further, when tested in a real-time place preference paradigm, SNI animals spent more time on the side exposed to amber light (which activates Arch), compared to the control side illuminated by blue light. And, sensory neurons isolated from SNI animals were more sensitive to mechanical probing, compared to control animals; this could be reversed by brief optogenetic inhibition of CGRPα soma.

Cowie and colleagues also tested if CGRPα neurons can drive inflammatory pain in an incision model of postoperative pain. In contrast to the SNI model, brief optogenetic inhibition was unable to reverse mechanical and heat hypersensitivity in the incision model. To investigate what drove the incisional pain, the researchers peripherally administered a CGRP peptide receptor antagonist, CGRP8-37, in the incision model. This attenuated mechanical hypersensitivity after three days of treatment, and immediately reversed the heat hypersensitivity observed in this model.

The researchers also showed that after incision, peripheral CGRPα was released from the CGRPα neurons, leading to CGRPα receptor signaling, which drives the inflammatory pain. This is in contrast to the neuropathic pain model, where peripheral peptide signaling is not involved, but rather it is the CGRPα neurons themselves that drive the pain.

In short, the work suggests differential roles for CGRPα-containing neurons, depending on the type of injury: CGRPα neurons themselves are essential for SNI-induced hypersensitivity, while CGRPα released from peripheral cutaneous CGRPα neurons is crucial for incision-induced inflammatory pain. This suggests that peripheral targeting of CGRPα-containing neurons will be more beneficial for neuropathic pain, and that peripheral targeting of the released peptide will be better for postsurgical inflammatory pain. The findings from this study are clinically relevant because CGRP ligand and CGRP receptor antibodies are having success in clinical trials of migraine; perhaps these drugs could be tried in other pain states as well.

Neuroimmune sex differences in pain

Sex differences in disease states are often understudied in preclinical laboratories. Many investigators in the pain field generally use male mice and rats, whereas the immunology field most often focuses on female rodents. The importance of using both males and females in preclinical research on connections between pain and the immune system was an important theme to emerge from pain presentations at SfN.

In a nanosymposium titled “Neuro-Immune Interactions in Pain, Migraine, and Itch,” Michael Burton, University of Texas at Dallas, US, presented work on the role of Toll-like receptor-4 (TLR4) present on macrophages in paclitaxel-induced neuropathic pain, and reported major sex differences in this regard. TLR4 is a member of the Toll-like receptor family and contributes to activation of the innate immune system.

It is well known that chemotherapy drugs such as paclitaxel can induce peripheral neuropathy and accompanying chronic pain that can last even after cessation of treatment. Previous studies have shown that paclitaxel increases rodent and human responses to TRPV1 agonists via activation of TLR4 (Li et al., 2015). However, because TLR4 is expressed on many different cell types, Burton sought to determine the specific TLR4-containing cell type that contributes to the development of paclitaxel-induced peripheral neuropathy.

The researchers used a transgenic approach in mice to delete TLR4 from macrophages; these animals are phenotypically identical to wild-type animals under normal conditions. Interestingly, Burton and colleagues found that when they gave paclitaxel to the macrophage-specific TLR4 knockouts, mechanical hypersensitivity was reversed in males, whereas females developed mechanical hypersensitivity similar to their wild-type littermates.

The researchers then delved into the mechanisms underlying these sex differences by examining macrophage phenotypes, including M1 macrophages, which promote pro-inflammatory responses, and M2 macrophages, which produce anti-inflammatory cytokines and regulate repair processes. Burton found that paclitaxel increased the M1 marker CD86 in wild-type males and females, and removal of macrophage-specific TLR4 significantly reduced this upregulation only in male animals. Meanwhile, the M2 marker, mannose receptor c-type (Mrc/CD206), was upregulated in both sexes in wild-type mice, and these levels were unchanged in both sexes of macrophage-specific TLR4 knockouts, in response to paclitaxel.

Burton also looked at the cell surface marker major histocompatibility complex (MHC), which is important in the acquired immune system’s recognition of foreign molecules. He found that paclitaxel increased MHC type II (MHCII)-positive macrophages in both wild-type male and females, but they were decreased in macrophage-specific TLR4 knockout males only.

Together, the data indicate that paclitaxel-induced neuropathic pain relies on TLR4 on macrophages in mice, but only in males. This research suggests that targeting TLR4 during chemotherapy treatment might be valuable in preventing pain. Furthermore, it raises the question of whether paclitaxel-induced neuropathic pain affects men more than women.

In a poster presentation, Candler Paige, University of Texas at Dallas, US, spoke of sex differences in hyperalgesic priming (a long-lasting hyper-responsiveness of pain-sensing neurons to inflammatory mediators) and neuropathic pain, making the case that CGRPα is important for the development and maintenance of both, but only in female mice and not males. CGRPα is released from nociceptors in response to tissue damage. Once released it causes vasodilation and inflammation in the tissue.

To test for sex differences in hyperalgesic priming and neuropathic pain, Paige and colleagues used a hyperalgesic priming paradigm in which the pro-inflammatory cytokine interleukin 6 (IL-6) is injected into the hindpaw to set up a “primed” state. Once the animals return to their baseline mechanical sensitivity, they receive intraplantar injections of the inflammatory molecule prostaglandin E2 (PGE2).

To assess CGRP involvement in hyperalgesic priming, the investigators administered the CGRP receptor antagonists olcegepant or CGRP8-37 intrathecally. When olcegepant was given at the time of IL-6 injection, the initial response to IL-6 was blocked in female but not male mice. When CGRP8-37 was administered with IL-6 treatment, the initial response to IL-6 as well as the hyperalgesic priming response to PGE2 were blocked in females only. Olcegepant or CGRP8-37 given at the time of PGE2 injection reversed hyperalgesic priming in female but not male mice.

The researchers also investigated the role of CGRP in the spared nerve injury (SNI) model of neuropathic pain and an incision model of inflammatory pain. In the former instance, CGRP8-37 decreased SNI-induced mechanical hypersensitivity in females but had no effect in males. In the latter case, when CGRP8-37 was administered at the time of incision, a decrease in mechanical hypersensitivity 24 hours after incision was observed in females only.

The findings suggest that CGRP receptor antagonists may relieve pain only in female but not male mice. The work also indicates that when testing CGRP-targeted therapies in clinical studies, researchers must treat males and females as separate cohorts, as sex-specific effects of the drugs may be diluted when both sexes are considered together.

Francie Moehring is a PhD candidate at the Medical College of Wisconsin, Milwaukee, US.